BILIRUBIN (TOTAL AND DIRECT)

BILIRUBIN (TOTAL AND DIRECT) BILIRUBIN (TOTAL) BILIRUBIN (DIRECT) BILIRUBIN (TOTAL) BILIRUBIN (DIRECT)
COD 11515 COD 11555 COD 11510 COD 11544 COD 11511 COD 11545
2 x 50 + 2 x 50 mL 500 + 500 mL 4 x 50 mL 2 x 500 mL 4 x 50 mL 2 x 500 mL
STORE AT 2-30ºC
Reagents for measurement of bilirubin concentration BILIRUBIN
Only for in vitro use in the clinical laboratory DIAZOTIZED SULFANILIC

PRINCIPLE OF THE METHOD In calculations of direct bilirubin, use the absorbance value obtained for the standard in the total bilirubin procedure
Direct bilirubin in the sample reacts with diazotized sulfanilic acid forming a coloured complex that can be measured by (Note 4).
spectrophotometry. Both direct and indirect bilirubin couple with diazo in the presence of cetrimide 1,2. The terms “direct” Mass concentration (mg/dL) x 17.1 = substance concentration (mol/L).
and “total” refer to the reaction characteristics of serum bilirubin in the absence or presence of solubilizing (accelerating)
reagents. The “direct” and “indirect” bilirubin are only approximately equivalent to the conjugated and unconjugated REFERENCE VALUES
fractions. Adults:

CONTENTS Total4: Up to 2.0 mg/dL = 34 mol/L

Reagent AT Reagent AD Reagent BT Reagent BD Direct5: Up to 0.3 mg/dL = 5 mol/L
COD 11515 2 x 40 mL 2 x 40 mL 2 x 10 mL 2 x 10 mL
COD 11555 400 mL 400 mL 1 x 100 mL 1 x 100 mL Newborns4 (total bilirubin) :
COD 11510 4 x 40 mL  4 x 10 mL  Age premature full-term
COD 11544 2 x 400 mL  2 x 100 mL 
Up to 24 h 1.0-8.0 mg/dL = 17-137 mol/L 2.0-6.0 mg/dL = 34-103 mol/L
COD 11511  4 x 40 mL  4 x 10 mL
Up to 48 h 6.0-12.0 mg/dL = 103-205 mol/L 6.0-10 mg/dL = 103-171 mol/L
COD 11545  2 x 400 mL  2 x 100 mL
3-5 days 10-14 mg/dL = 171-239 mol/L 4.0-8.0 mg/dL = 68-137 mol/L
COMPOSITION
These ranges are given for orientation only; each laboratory should establish its own reference ranges.
BILIRUBIN (TOTAL)
QUALITY CONTROL
AT. Reagent. Sulfanilic acid 29 mmol/L, hydrochloric acid 0.2 mol/L, cetrimide 50 mmol/L.
DANGER: H314: Causes severe skin burns and eye damage. P280: Wear protective gloves/protective It is recommended to use the Biochemistry Control Serum level I (cod. 18005, 18009 and 18042) and II (cod. 18007,
clothing/eye protection/face protection. P303+P361+P353: IF ON SKIN (or hair): Remove/Take off immediately 18010 and 18043) to verify the performance of the measurement procedure.
all contaminated clothing. Rinse skin with water/shower. Each laboratory should establish its own internal Quality Control scheme and procedures for corrective action if controls
BT. Reagent. Sodium nitrite 11.6 mmol/L. do not recover within the acceptable tolerances.
BILIRUBIN (DIRECT) METROLOGICAL CHARACTERISTICS
AD. Reagent. Sulfanilic acid 35 mmol/L, hydrochloric acid 0.24 mol/L.
 Detection limit (Total bilirubin): 0.003 mg/dL = 0.05 mol/L
DANGER: H314: Causes severe skin burns and eye damage. P280: Wear protective gloves/protective
clothing/eye protection/face protection. P303+P361+P353: IF ON SKIN (or hair): Remove/Take off immediately  Detection limit (Direct bilirubin): 0.02 mg/dL = 0.34 mol/L
all contaminated clothing. Rinse skin with water/shower.  Linearity limit: 20 mg/dL = 343 mol/L. For higher values dilute sample 1/3 with distilled water and repeat
BD. Reagent. Sodium nitrite 3.5 mmol/L. measurement.
For further warnings and precautions, see the product safety data sheet (SDS).  Repeatibility (within run):

STORAGE Total bilirubin CV n Direct bilirubin CV n

Store at 2-30ºC. 0.59 mg/dL = 10.1 mol/L 3.0 % 20 0.77 mg/dL = 13.2 mol/L 1.2 % 20
Reagents are stable until the expiry date shown on the label when stored tightly closed and if contaminations are 6.74 mg/dL = 115.2 mol/L 1.0 % 20 1.36 mg/dL = 23.2 mol/L 0.5 % 20
prevented during their use.
Indications of deterioration:  Reproducibility (run to run):
 Reagents: Presence of particulate material, turbidity, absorbance over 0.05 at 540 nm (1 cm cuvette). Total bilirubin CV n Direct bilirubin CV n
AUXILIARY REAGENTS 0.59 mg/dL = 10.1 mol/L 3.6 % 25 0.77 mg/dL = 13.2 mol/L 2.3 % 25
S. Bilirubin Standard (cod 11513). Reconstitute with 5.0 mL of distilled water (Note 1). Concentration is given on the 6.74 mg/dL = 115.2 mol/L 3.3 % 25 1.36 mg/dL = 23.2 mol/L 0.9 % 25
label. Concentration value is traceable to the Standard Reference Material 916a (National Institute of Standards and
Technology, USA). Protect the reconstituted Standard from light. Stable for 4 hours at 15-30ºC or for 2 months at –  Sensitivity (total bilirubin): 88 mAdL/mg = 5.15 mAL/mol
18ºC when frozen in aliquots.  Sensitivity (direct bilirubin): 100 mAdL/mg = 5.85 mAL/mol
REAGENT PREPARATION  Trueness: Results obtained with this reagent did not show systematic differences when compared with reference
reagents (Note 4). Details of the comparison experiments are available on request.
Working Reagent: Transfer the contents of one Reagent BT vial into a Reagent AT bottle for total bilirubin
 Interferences: Hemoglobin (10 g/L) does not interfere. Lipemia (triglycerides > 15 g/L) may intefere. Other drugs
determination, or one Reagent BD vial into a Reagent AD bottle for direct bilirubin determination (Note 2). Mix
and substances may interfere6.
thoroughly. Other volumes can be prepared in the proportion: 1 mL Reagent BT + 4 mL Reagent AT or 1 mL Reagent
BD + 4 mL Reagent AD. Stable for 20 days at 2-8ºC. These metrological characteristics have been obtained using an analyzer. Results may vary if a different instrument or a
manual procedure are used.
ADDITIONAL EQUIPMENT
DIAGNOSTIC CHARACTERISTICS
 Analyzer, spectrophotometer or photometer with cell holder thermostatable at 37ºC and able to read at 540  20 nm.
Bilirubin is a waste product derived from the heme moiety of the hemoglobin released from senescent or damaged
 Cuvettes with 1 cm light path (if factor is used in calculations). erythrocytes, that are destroyed in the reticuloendothelial cells. After production, bilirubin is transported to the liver in
SAMPLES association with albumin. Inside the hepatocytes bilirubin is conjugated with glucuronic acid and it is excreted into bile.
A number of inherited and acquired diseases affect production, uptake, metabolism, and excretion of bilirubin, resulting
Serum collected by standard procedures. in hyperbilirubinemia4,7.
Bilirubin in serum is stable for 7 days at 2-8ºC if serum is protected from light3. Unconjugated hyperbilirubinemia is seen in newborns (physiological jaundice), in increased red cell destruction
(hemolytic anemia, extensive hematoma), in ineffective erythropoiesis and in some rare genetic diseases (Gilbert’s
PROCEDURE FOR TOTAL BILIRUBIN syndrome, Crigler-Najjar syndrome).
1. Pipette into labelled test tubes: (Notes 1, 3) Conjugated hyperbilirubinemia is associated to a decreased excretion of bile due to liver diseases (hepatitis or cirrhosis)
or to intrahepatic or extrahepatic cholestasis.
Reagent Blank Sample Blank Sample Standard
Jaundice is a clinical manifestation of hyperbilirubinemia, consisting of deposition of bile pigments in the skin, resulting
Distilled water 100 µL    in a yellowish staining of the skin and mucous membranes.
Sample  100 µL 100 µL  Clinical diagnosis should not be made on the findings of a single test result, but should integrate both clinical and
Standard (S)    100 µL laboratory data.
Reagent (AT)  1.0 mL   NOTES
Working Reagent 1.0 mL  1.0 mL 1.0 mL
1. For bilirubin determination in newborns, reconstitute the Standard with 1.0 mL of distilled water. The Standard
2. Mix thoroughly and let stand the tubes for 2 minutes at room temperature. concentration will be that stated on the vial label multiplied by 5. Reduce sample volume (water, standard, serum) to
3. Read the absorbance (A) of the Sample Blanks at 540 nm against distilled water. 50 L and use the concentrated Standard. Method linearity is then doubled (up to 40 mg/dL = 686 mol/L).
2. It is advisable to wash the Reagent B vial with a small volume of the prepared mixture in order to completely rinse
4. Read the absorbance (A) of the Samples and of the Standard at 540 nm against the Reagent Blank.
the vial and avoid any losses.
PROCEDURE FOR DIRECT BILIRUBIN 3. These reagents may be used in several automatic analysers. Instructions for many of them are available on
1. Pipette into labelled test tubes: (Notes 1, 3). request.
4. Calibration with the provided aqueous standard may cause a matrix related bias, specially in some analyzers. In
Reagent Blank Sample Blank Sample these cases, it is recommended to calibrate using a serum based standard (Biochemistry Calibrator, cod. 18011
and 18044).
Distilled water 100 µL  
Sample  100 µL 100 µL BIBLIOGRAPHY
Reagent (AD)  1.0 mL  1. Pearlman FC and Lee RTY. Detection and measurement of total bilirubin in serum, with use of surfactants as
Working Reagent 1.0 mL  1.0 mL solubilizing agents. Clin Chem 1974; 20: 447-453.
2. Zoppi F, Peracino A, Fenili D, Marcovina S and Ramella C. Metodo per la determinazione della bilirubina totale e
2. Mix thoroughly and let the tubes stand for exactly 5 minutes at 37ºC.
coniugata. Uso di un tensioattivo cationico come agente solubilizzante. Giorn It Chim Cl 1976; 1:343-359.
3. Read the absorbance (A) of the Sample Blanks at 540 nm against distilled water.
3. World Health Organization (WHO). Use of anticoagulants in diagnostic laboratory investigations. Document
4. Read the absorbance (A) of the Samples at 540 nm against the Reagent Blank. WHO/DIL/LAB/99.1, Rev.2; 2002.
CALCULATIONS 4. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics, 4th ed. Burtis CA, Ashwood ER, Bruns DE. WB
Saunders Co, 2005.
The bilirubin concentration in the sample is calculated using the following general formula: 5. McPherson RA, Pincus MR. Henry's Clinical Diagnosis and Management by Laboratory Methods. 20st ed.
A Sample – A Sample Blank Saunders Elsevier, 2001:1427
x C Standard = C Sample 6. Young DS. Effects of drugs on clinical laboratory tests, 5th ed. AACC Press, 2000.
A Standard
7. Friedman and Young. Effects of disease on clinical laboratory tests, 4th ed. AACC Press, 2001.

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